1. Field of the Invention (Technical Field)
The present invention relates to the in vivo tagging of leukocytes, and in particular lymphocytes, with radioactive tracers and other diagnostically useful metal ions, and subsequent detection of lymphocyte trafficking and sites of concentrated lymphocytes within the mammal by radiodetection or other means.
2. Description of the Related Art, Including Information Disclosed under 37 C.F.R. Sections 1.97-1.99 (Background Art)
This application covers generally the development of a pan-T-lymphocyte tracer that can be used both to study trafficking of stimulated lymphocytes and as a diagnostic radiopharmaceutical or magnetic resonance imaging diagnostic pharmaceutical, for use in chronic infections such as osteomyelitis and granulomatous diseases and for other conditions. One of the disadvantages of using only monoclonal antibody tracers to tag circulating lymphocytes is that they are generally species specific, and a monoclonal antibody that recognizes a specific subset of human lymphocytes can be used only for studies in humans. Thus, there is a need for specific tracers that can be used in both laboratory animals and man, so that experimental studies, the results of which will be extrapolated to humans, can be conducted.
One class of pan-T-lymphocyte tracers disclosed herein are leukostimulatory lectins, and particularly the plant-derived lectin, phytohemagglutinin-L4 (PHA-L4). This isolectin binds the CD3 receptor on T-lymphocytes of both laboratory animals and humans. Wimer, B. M., "The ideal biological response modifier," Mol Biother 1:311-317, 1989. It also stimulates the lymphocytes to differentiate and divide (Wimer, B. M., "Characteristics of PHA-L4, the mitogenic isolectin of phytohemagglutinin, as an ideal biologic response modifier," Mol Biother 2:4-17, 1990); thus, the labeled molecule provides a tracer that can be used to track stimulated lymphocytes.
Although the use of PHA as a drug has been studied for many years (See generally, Wimer, B. M., "Therapeutic activities of PHA-L4, the mitogenic isolectin of phytohemagglutinin," Mol Biother 2:74-90, 1990; and, Wimer, B. M., "Potential therapeutic applications of PHA-L4, the mitogenic isolectin of phytohemagglutinin," Mol Biother 2:196-200, 1990), it has not found a permanent place in the routine practice of medicine.
Native PHA consists of four subunits and has both leuko- and erythro-agglutinating properties, as well as leukostimulatory properties. The L4 isolectin of PHA carries only the leukoagglutinating and stimulating species, and has been found to bind the CD3 receptor on T-lymphocytes. Greaves, M. F., Bauminger, S., Janossy, G., "Lymphocyte activation. III. Binding sites for phytomitogens on lymphocyte subpopulations," Clin Exp Immunol 10:537-554, 1972.
PHA was originally isolated as an aqueous extract of the beans of the genus Phaseolus, especially the red kidney bean, Phaseolus vulgaris. At least two active ingredients have been identified in the extract, a mucoprotein and a glycoprotein. The L-4 isolectin appears to be one of five isolectins that comprise the PHA glycoprotein. PHA may be prepared by a number of means, and is commercially available from E-Y Laboratories in a variety of forms, including the glycoprotein tetramer and the purified L-4 isolectin of PHA.
PHA-L4 may also be prepared using recombinant DNA technology. Hoffman and Donaldson have reported the methodology for synthesis of non-erythroagglutinating PHA-L4 in Escherichia coli. Hoffman, L. M., and Donaldson, D. D., "Synthesis of mitogenic phytohemagglutinin-L in Escherichia coli," Biotechnology 5:157-160, 1987; Hoffman, L. M., U.S. Pat. No. 4,870,015, Method and Composition for Producing Lectin in Microorganisms.
One advantage of using labeled PHA-L4 or other lectin-based leukostimulatory agents in place of monoclonal antibody tracers is the possibility of using the leukostimulatory agent, such as PHA-L4 isolectin, in several species. Generally speaking, monoclonal antibodies against the human CD3 receptor do not bind to murine CD3 receptors, and this lack of a lymphocyte tracer applicable to the laboratory mouse has limited many basic studies of adoptive immunotherapy.
Rosenberg (Rosenberg, S., "Lymphokine-activated killer cells: A new approach to immunotherapy of cancer," JNCI 75:595-603, 1985) notes that immunologically active cells tend to be larger than normal resting cells. They are also different in other aspects, such as their level of oxidative metabolism, degranulation, and adherence. e1-Hag, A., Clark, R. A., "Immunosuppression by activated human neutrophils. Dependence on the myeloperoxidase system," J Immunol 139:2406-2413, 1987. Thus, it appears that cells that are stimulated and labeled with radiolabeled PHA-L4 will traffic differently than the general population of lymphocytes, which can be labeled by methods such as .sup.111 In oxine. Thakur, M. L., Coleman, R. E., Welch, M. J., "Indium-111-labeled leukocytes for the localization of abscesses: preparation, analysis, tissue distribution, and comparison with gallium-67 citrate in dogs," J Lab Clin Med 89:217-228, 1977; and, Thakur, M. L., Lavender, J. P., Arnot, R. N., et al, "Indium-111-labeled autologous leukocytes in man," J Nucl Med 18:1014-1021, 1977.
In vivo imaging with .sup.111 In-labeled lymphocytes in a study of patients with Hodgkin's disease was demonstrated by Lavender et al in 1977. Lavender, J. P., Goldman, J. M., Arnot, R. N., Thakur, M. L., "Kinetics of indium-111 labeled lymphocytes in normal subjects and patients with Hodgkin's disease," Brit Med J 2:797-799, 1977. Such studies have been limited by the extreme radiosensitivity of lymphocytes labeled internally with .sup.111 In. A more recent approach has been to use radiolabeled antibodies that bind to cell surface antigens to label the cells in such a way that the radionuclide is distanced from the cell nucleus. Thakur, M. L., U.S. Pat. No. 4,917,878, Novel Use of a Radiolabelled Antibody Against Stage Specific Embryonic Antigen for the Detection of Occult Abscesses in Mammals; Houston, L. L., Nowinski, R. C., Bernstein, I. D., "Specific in vivo localization of monoclonal antibodies directed against the Thy 1.1 antigen," J Immunol 125:837-843, 1980; and, Loutfi, I., Chisholm, P. M., Bevan, D., Lavender, J. P. "In vivo imaging of rat lymphocytes with an indium 111-labeled anti-T cell monoclonal antibody: a comparison with indium-111-labeled lymphocytes," Eur J Nucl Med 16:69-76, 1990.
Labeled lymphocytes are useful for studying lymphocyte trafficking and for diagnostic imaging of chronic infections and granulomas, and some tumors. Wagstaff, J., Gibson, C., Thatcher, N., et al, "A method for following human lymphocyte traffic using Indium-111 oxine labeling," Clin Exp Immunol 43:443-449, 1981; and, Thakur, M. L., "A look at radiolabeled blood cells," Nucl Med Biol 13:147-158, 1986.
The use of antibodies for in vivo tagging of leukocytes is known in the art. Goodwin, D. A., and Meares, C. F., U.S. Pat. No. 4,634,586, Reagent and Method for Radioimaging Leukocytes, teach a method in which leukocytes are radioimmunoimaged by injecting patients with an immunoreactive nonleukocidal conjugate of an anti-leukocyte and a gamma-emitting radioactive metal chelate, waiting for the conjugate to localize on the leukocytes, injecting the patient with an antibody to the conjugate to clear the blood of background nonlocalized conjugate and visualizing the leukocytes by scintillation scanning. The method can also be used without the step of injecting the second antibody to clear background nonlocalized antibody. Thakur, M. L., U.S. Pat. No. 4,917,878, Novel Use of a Radiolabelled Antibody Against Stage Specific Embryonic Antigen for the Detection of Occult Abscesses in Mammals, teaches a method whereby antibodies against a particular antigen found on human granulocytes, stage specific embryonic antigen-1, are radiolabeled using a bifunctional chelating agent, and the resulting radiolabeled antibody reagent injected under conditions which allow the reagent to accumulate at sites of occult abscess.
The use of radioisotopes to label biologically derived substances is well known. These compositions can be used in assays, can be administered to the human body to visualize or monitor functioning of various parts of the body, and can be used for therapy. A variety of radioisotopes, including isotopes of iodine, technetium, indium, gallium, yttrium and rhenium have been used.
Different methods can be used to radiolabel biological substances with radioisotopes. For iodine, a variety of iodination methods, such as chloramine-T, iodine monochloride, enzymatic iodination, electrolytic procedures and conjugation labeling, are well recognized. (Pettit, W.A., et al, "Iodination and acceptance testing of antibodies," Tumor Imaging: The Radioimmunochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, Eds., Masson Publishing USA Inc., New York, 1982, pp 99-109.) Bifunctional chelate methods, such as DTPA conjugation, can be used to label antibodies with .sup.99m Tc, .sup.111 In, .sup.67 Ga, .sup.68 Ga or similar radionuclides. (Wensel, T. G. and Meares, C. F., "`Bifunctional` Chelating Agents for Binding Metal Ions to Proteins," Radioimmunoimaging and Radioimmunotherapy, S W Burchiel and B A Rhodes, eds., Elsevier Publishing Co., New York, 1983, pp 185-196.) The bifunctional chelate method was introduced by Krejcarek, G. E. and Tucker, K. L. (Biophys Res Comm 77:581-585, 1977) and has been widely employed in many variations using a broad variety of bifunctional chelating agents.
U.S. Pat. No. 4,479,930 to Hnatowich, D. J., discloses methods of radiolabeling using a dicyclic dianhydride of compounds such as ethylenediaminetetraacetic acid or diethylenetriaminepentaacetic acid. The patent also discloses chemical compositions containing the chelating agents and proteins or polypeptides. U.S. Pat. No. 4,668,503 to Hnatowich, D. J., discloses a process for labeling amines with .sup.99m mTc in the presence of a stannous reducing agent. U.S. Pat. No. 4,622,420 to Meares, C. F., et al, discloses chelating agents which are analogs of ethylenediaminetetraacetic acid, ethylenediaminatriacetic acid or ethylenediaminepentaacetic acid which are useful in attaching radiolabels to biological molecules. Numerous other methods of labeling proteins and like substances which include lysine-containing amino acid groups, including those disclosed by Haber, E., and Khaw, B. A., U.S. Pat. No. 4,421,735; and by Fritzberg, A. R., and Kasina, S., U.S. Pat. No. 4,670,545, and by Baidoo, K. E., et al, ".sup.99m Tc Labeling of Proteins: Initial Evaluation of Novel Diaminedithiol Bifunctional Chelating Agent," Cancer Res (Supp) 50:799s-803s, 1990, are well known in the art. A review article by Fritzberg et al discusses the general bifunctional chelate methods which may be used (Fritzberg, A. R., Berninger, R. W., Hadley, S. W., and Wester, D. W., "Approaches to radiolabeling of antibodies for diagnosis and therapy of cancer," Pharm Res 5:325-334, 1988).
Another general approach is direct labeling, which works with antibodies and other proteins containing accessible disulfide bonds or monosulfides. Although several direct methods have been reported, the first direct method capable of providing a sufficiently strong bond between the protein and the .sup.99m Tc for in vivo applications was the direct or pretinning method described in U.S. Pat. No. 4,424,200, entitled Method for Radiolabeling Proteins with Technetium-99m, to Crockford, D. R., and Rhodes, B. A. In this method, a single reduction compound, consisting of stannous [Sn(II)] chloride and other salts which serves both to reduce the protein, thereby exposing the disulfide bonds, and to reduce the sodium pertechnetate, is used. With this method, many proteins can be successfully radiolabeled with .sup.99m Tc. In U.S. Pat. No. 5,078,985, entitled RadiolabelingAntibodies and Other Proteins with Technetium or Rhenium by Regulated Reduction, to Rhodes, B. A., a method is provided in which any of a variety of reducing agents can be used to reduce disulfide bonds, the reducing agent is then removed, a source of Sn(II) added, and the preparation then labeled.
Other methods for direct labeling have been reported on (Schwarz, A., and Steinstruaber, A., "A novel approach to Tc-99m-labeled monoclonal antibodies," J Nucl Med 28:721, 1987; Pak, K. Y., et al, "A rapid and efficient method for labeling IgG antibodies with Tc-99m and comparison to Tc-99m Fab'". J Nucl Med 30:793, 1989; Granowska, M., et al, "A Tc-99m-labeled monoclonal antibody, PR1A3, for radioimmunoscintigraphy," J Nucl Med 30:748, 1989; Reno, J. W., U.S. Pat. No. 4,877,868, Radionuclide Antibody Coupling). In the equivalent methods disulfide reducing agents other than stannous salts were used. Pak et al used dithiothreitol to reduce the disulfide bonds of the antibody; Swartz and Steinsbruaber, and Granowska et al used 2-mercaptoethanol; Reno used dithiothreitol (DTT) to reduce the disulfide groups of the protein, then protected the reactive sulfides with Zn (II) or other sulfhydryl group derivatizing reagents. Also some of these investigators (Swartz and Steinsbruaber, and Granowska et al) reduced the .sup.99m Tc prior to adding it to the reduced antibody. The review by Rhodes (Rhodes, B. A., "Direct Labeling of Proteins with .sup.99m Tc," Nucl Med Biol 18:667-676, 1991) covers direct labeling methods in detail, while the reviews of Hnatowich generally cover radiolabeling methods (Hnatowich, D. J., "Antibody radiolabeling, problems and promises," Nucl Med Biol 17:49-55 (1990); and, Hnatowich, D. J., "Recent developments in the radiolabeling of antibodies with iodine, indium, and technetium," Semin Nucl Med 20:80-91, 1991).